Radiosynthesis and biological evaluation of two 18F-labeled PET radioligands for α-synuclein

Introduction: The aggregation of misfolded α-synuclein protein is a pathological hallmark of Parkinson’s disease (PD), Dementia with Lewy Bodies (DLB) and Multiple System Atrophy (MSA). In vivo imaging of α-synuclein aggregations with a clinically suitable positron emission tomography (PET) radioligand could provide important information in the early diagnosis of PD, disease progression, and assessment of the efficacy of disease-modifying therapies. We report here the ¹⁸F radiolabeling and evaluation of two radioligands, [¹⁸F]TZ83-41 and [¹⁸F]TZ80-156, as potential imaging agents for α-synuclein aggregation.

Methods: The radiosynthesis of [¹⁸F]TZ83-41 was accomplished by a two-step procedure with [¹⁸F]fluoroethyl ptoluenesulfonate as a prosthetic group, followed by O-alkylation to the quinolinyl-containing phenol group. [¹⁸F]TZ80-156 was synthesized starting from the allylic bromide precursor via nucleophilic halogen exchange (halex) reaction with [¹⁸F]fluoride, followed by deprotection of the N-Boc amine with trifluoroacetic acid. Homologous competitive binding assays with α-synuclein fibrils amplified from PD tissue were used to determine the direct binding affinity of the two radioligands. The biodistribution study was performed in male Sprague Dawley (SD) rats at four time points (5, 30, 60 min and 120 min) after intravenous injection of the radiotracer. The uptake of radioactivity was calculated as percentage injected dose per gram (%ID/gram). Both radiotracers were evaluated with microPET imaging using a Focus 220 microPET scanner to collect dynamic scans after intravenous injection into male macaques (NHP).

Results: The radioligands [¹⁸F]TZ83-41 and [¹⁸F]TZ80-156 were successfully produced in 17 ± 3% (n = 2) and 25 ± 5% (n = 2) radiochemical yield (RCY) respectively with high molar activity (> 37 GBq/μmol) and high radiochemical purity (> 99%), at the end of synthesis (EOS). Direct radioligand binding assays showed [¹⁸F]TZ83-41 and [¹⁸F]TZ80-156 have good to moderate binding affinity for α-synuclein fibrils (7.6 nM and 35 nM, respectively), as well as little to no binding to Alzheimer’s disease brain tissue homogenate (Data not shown). MicroPET imaging revealed the brain standardized uptake value (SUV) for [¹⁸F]TZ83-41 and [¹⁸F]TZ80-156 reached a maximum (~2.0) at 6-7 min post tracer injection, followed by a favorable washout rate from the NHP brain. Biodistribution studies indicated that [¹⁸F]TZ83-41 had a good brain uptake up to 0.71~0.24 %ID/gram from 5 to 120 min. For [¹⁸F]TZ80-156, the initial brain uptake (%ID/gram) was high with 0.86 at 5 min, but dramatically decreased to 0.25 at 30 min, and 0.16 at 60 min. Significant defluorination was observed in [¹⁸F]TZ80-156 since bone uptake was increased from 0.45 at 5 min to 2.18 at 30 min and 3.09 at 120 min.

Conclusions: Two new ¹⁸F labeled radioligands,[¹⁸F]TZ83-41 and [¹⁸F]TZ80-156 were radiosynthesized and characterized. Both radiotracers crossed the blood brain barrier and possessed favorable washout kinetics in the NHP brain. Direct radioligand binding assays indicated higher binding affinity of [¹⁸F]TZ83-41 compared to [¹⁸F]TZ80-156 for α-synuclein fibrils. Defluorination of [¹⁸F]TZ80-156 makes [¹⁸F]TZ83-41 the better candidate for future translational investigations. Additional radiometabolism studies and characterization in corresponding disease models are warranted for clinical applications of this promising radiotracer.

Research Support: This work was supported by the National Institutes of Health (U19 NS110456).

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